The mammalian sirtuins (SIRT1-7) are NAD+-dependent deacetylase enzymes that deacetylate cellular proteins to regulate their target proteins. Sirtuins are activated by nutritional stress, exemplified by low calorie diets, and are thought to be important signal transducers that adapt cells and human physiology. There is considerable interest in understanding how sirtuins are regulated on a molecular level. Such understanding could provide opportunities to better target them for pharmacologic modulation. A key limitation in current technology is the inability to directly assess sirtuin activity in the cel. The ability to determine the activity of a specific sirtuin in a cell at a given time, under conditons of a defined perturbation would significantly accelerate investigation into sirtuin regulation. For example, current models indicate that some sirtuins are regulated by changes in NAD+ metabolism (including NAD+ or NAD+/NADH ratio). Because sirtuin activity cannot be directly measured in cells, it is not possible to verify this idea directly, but only by inference through deacetylation of target proteins. Such an approach is subject to many confounding effects. Another key question in the sirtuin field is whether activation of signaling pathways initiated by insulin or glucagon affects sirtuin activity. Recent data suggests that cAMP signaling increases SIRT1 activity, but this activity change cannot be shown directly in cells. Finally, key questions in the field relate to pharmacological activators such as resveratrol. Does resveratrol cause SIRT1 activity to increase in cells? This question is of considerable importance in the sirtuin fied and has remained unresolved since 2003. This grant application proposes development of a breakthrough technology in being able to image sirtuin activity in cells via an activity-based probe. The probe incorporates a clickable group, which enables conjugation of the probe to a dye to provide visualization. Our preliminary data establishes that the probe is cell permeable, that probe labeling of sirtuins requires the enzyme mechanism, that probes are sensitive to cellular NAD+ levels, and that time-dependence of labeling can be used as a measure of sirtuin activity in cells. We propose to develop the probe and use it to address key questions of interest to the sirtuin field. To do so we propose the following specific aims: In Aim 1 we will investigate the concentration and time dependencies of labeling of SIRT1-7 in cells, thereby providing a determination of probe properties against each sirtuin isoform. In Aim 2, we will address the role of NAD+ metabolism in altering SIRT1-7 activities in cells and address key issues, such as effect of NAD+ level and NAD+/NADH ratio in affecting sirtuin activity. In Aim 3: we will screen agonists of cAMP signaling, AMP kinase signaling, and insulin and glucagon for their affects on sirtuin activity. We will also address the ability of inhibitors and activators to alter sirtuin acivities in cells. The wider availability of our tools will accelerate investigations of sirtuins by other researchers and provide stimulus to extend these strategies to study other key signaling processes.